Two Toxins from Conus striatus That Individually Induce Tetanic Paralysis

Abstract

We describe structural properties and biological activities of two related O-glycosylated peptide toxins isolated from injected (milked) venom of Conus striatus, a piscivorous snail that captures prey by injecting a venom that induces a violent, spastic paralysis. One 30-amino-acid toxin is identified as κA-SIVA (termed s4a here), and another 37-amino-acid toxin, s4b, corresponds to a putative peptide encoded by a previously reported cDNA. We confirm the amino acid sequences and carry out structural analyses of both mature toxins using multiple mass spectrometric techniques. These include electrospray ionization ion trap mass spectrometry and nanoelectrospray techniques for small volume samples, as well as matrix-assisted laser desorption/ionization time-of-flight mass spectrometric analysis as a complementary method to assist in the determination of post-translational modifications, including O-linked glycosylation. Physiological experiments indicate that both s4a and s4b induce intense repetitive-firing of the frog neuromuscular junction, leading to a tetanic contracture in muscle fiber. These effects apparently involve modification of voltage-gated sodium channels in motor axons. Notably, application of either s4a or s4b alone mimics the biological effects of the whole injected venom on fish prey.Toxic components of Conus venoms are predominately small peptides (12-50 amino acids), most of which are extensively cross-linked with internal disulfide bonds that add rigidity and stability to the peptide and constrain its conformation for optimal interaction with specific receptors. In addition to disulfide linkages, cone snail peptides have an unprecedented density and diversity of other amino acid modifications that occur after translation of the peptide chain. It is likely that many of these post-translational modifications (PTMs) also confer specificity to the peptide toxins, which can be highly selective for certain receptor subtypes (1,2). For *To whom correspondence should be addressed. E-mail: sweedler@scs.uiuc.edu. Tel: (217) NIH Public Access

Glycosylation of Conotoxins

“…The nature of the monosaccharides and type of linkages were not determined. Later, the Hex 3 HexNAc 2 glycan moiety (893 Da) of κA-SIVA (s4a) was confirmed by Jakubowski et al [55] using LC/ESI-MS, and by Kelley et al [53] using MALDI-TOF-MS and LC/ESI-MS. Evidence was presented for the occurrence of a HexNAc-HexNAc fragment, to which three Hex residues are connected.…”

“…The mature neurotoxic conopeptide κA-conotoxin SIVA (κA-SIVA, also called s4a) isolated from its venom has been shown to be active on tetrodotoxin-sensitive voltage-gated sodium (Na v ) channels [53]—though not on voltage-gated potassium channels as thought earlier [54]—thus eliciting spastic paralytic symptoms when injected into the fish during prey capture. The conotoxin induces intense repetitive firing of the frog neuromuscular junction leading to a tetanic contracture in muscle fiber [53]. It has a backbone of 30 amino acids with pyroglutamic acid at the N -terminal site, three 4- trans -hydroxyprolines, amidated cysteine at the C -terminal side, and three disulfide bonds.…”

Diversity of conotoxin types from Conus californicus reflects a diversity of prey types and a novel evolutionary history

“…Disulfide linkages were reduced by treating part of the venom sample with dithiothreitol, and the resulting free sulfhydryl groups were alkylated with iodoacetamide. After purification, this material was subjected to capillary HPLC with analysis in parallel by electrospray ionization and MALDI MS, in general as described previously (Jakubowski et al, 2006; Kelley et al, 2006). For full details see Mass Spectral Analysis, Supplementary Material.…”

Investigating Endogenous Peptides and Peptidases Using Peptidomics

“…Many more examples can be presented as bioactive peptides can be found in many biological niches and play many roles. Conotoxins, for example, are a group of neurotoxic peptides produced by the venomous marine cone snail that are potential therapeutics (15-18). Here, we primarily focus on the application of peptidomics to problems in mammalian biology, the exception being the work described on honeybee neuropeptidomics, but we recognize that peptidomics has the potential to impact many different areas of biology.…”